Tyrosine phosphorylation and dephosphorylation of proteins is known to play a central role in regulation of cell growth and differentiation. Activation of resting T or B lymphocytes via their antigen-specific receptors TCR and BCR, respectively) or NK cells through their type III Fcg receptor (CD16), is initiated by rapid tyrosine phosphorylation of a number of regulatory proteins, such as phospholipase Cgl and proteins that bind phosphatidylinositol 3-kinase (PI3K). Identification of the protein tyrosine kinases (PTKs) involved, their regulators and their substrates, is important to our understanding of lymphocyte physiology in health and disease. During the last few years, it has become clear the PTKs of both the Src and Syk families play crucial roles in this signaling. The objective of this competitive renewal is to continue to study the regulation of the catalytic activity of the two Syk family PTKs present in lymphoid cells, Syk and Zap, and investigate their roles in the regulation of tyrosine phosphorylation in these cells. Specific Aim 1 addresses the role and regulation of Syk and Zap in lymphoid cells. The investigator proposes to build on his advances and continue with more indepth studies as well as experiments addressing the physiological significance of his observations. He anticipates learning what the role, importance, and targets of the Syk family PTKs Syk and Zap are in lymphoid cells, and whether the differential biochemical properties between these two PTKs is of biological importance. Specific Aim 2 investigates the regulation of SH2 domains by tyrosine phosphorylation, a major discovery supported by this grant. This novel Syk-and Zap-mediated regulation of Lck will be studied in more detail, and he will examine whether it is a more general mechanism in signal transduction. Specific Aim 3 examines the role of phosphatidylinositol 3-kinase (Pl3K) in Syk- and Zap-mediated signaling. He has reported that PI3K plays an important and complex role downstream of Syk and Zap in the MAP kinase pathway and in the activation of the-NFAT element of the IL-2 gene. He will extend these studies and attempt to define the biochemical steps between Syk or Zap and P13K in TCR signaling and to elucidate the regulation of MAP kinases by P13K in detail. Overall, the proposed studies will provide a detailed characterization of the function and substrates of the Syk family kinases, which seem to play an important role in T and B cell activation in concert with the Src family kinases and other key regulatory enzymes.